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Biomedical subjects

D E Lopatin

Publications and source records attributed to D E Lopatin.

At least 19 recordsLinked to original sources
PubMed1997

Occurrence of Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola in periodontally healthy and diseased subjects as determined by an ELISA technique.

The aim of this study was to assess by means of an ELISA technique, the occurrence of 3 putative periodontopathogens, Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola, in 3 clinically-defined adult periodontal conditions. Thirty systemically-healthy subjects were selected and grouped into 3 categories according to their periodontal health: 1) 10 periodontitis subjects (PS), having moderate adult chronic periodontitis; 2) 10 untreated gingivitis subjects (UGS), exhibiting no signs of periodontal destruction but presenting with clinical signs of mild gingivitis; and, 3) 10 treated gingivitis subjects (TGS), having the same clinical status as UGS, but who received a thorough prophylaxis treatment within the past 7 to 14 days prior to the baseline examination. A total of 60 samples were collected subgingivally from the six Ramfjord teeth per subject in each group and ELISA analysis was carried out to give a semiquantitative estimate of P. gingivalis. B. forsythus, and T. denticola. The immunologic detection method suggested the presence of antigens of P. gingivalis, B. forsythus, and T. denticola in subjects from each of the 3 groups. When a global analysis for the 3 disease groups was performed at one time, statistically significant differences were found among the ELISA scores of the 3 bacterial species. For example, comparisons of the ELISA scores showed that the concentrations of P. gingivalis differed significantly when comparing TGS to UGS and PS, but not when examining UGS/PS. The ELISA scores for B. forsythus were significantly different between TGS and PS. Mean concentrations of T. denticola were significantly different when comparing PS to TGS or UGS, whereas no difference was found between the latter categories. Within the limited scope of this study, the concentration of antigens detectable from putative periodontopathogens like P. gingivalis, B. forsythus, and T. denticola differed among the 3 diseased groups, with periodontitis subjects often showing the greatest level of antigens. Thus, it is reasonable to expect that, when using sensitive immunological detection methods, antigens of suspected periodontal pathogens can be found irrespective of the individual's clinical status. However, while detectable in the periodontal sites, the concentrations of these microorganisms are most likely to be above the threshold necessary to induce clinically-significant disease. Studies with larger sample size and standardized antigens are necessary to determine if the groups we found not to differ, were, in fact, different.

Adult↗
PubMed1997

Bacterial adherence to guided tissue regeneration barrier membranes exposed to the oral environment.

Microbial colonization of barrier materials used in guided tissue regeneration (GTR) is known to adversely affect treatment outcomes. The purpose of this study was to compare the rate at which 11 commonly-occurring oral bacteria species colonize three different barrier materials (collagen, expanded polytetrafluoroethylene, and polylactic acid). The study group consisted of 10 systemically healthy individuals with no history of periodontal disease and absence of antimicrobial therapy within the previous 3 months. In each patient, 4 teeth per quadrant (P1, P2, M1, M2) were selected and 3 teeth were randomly assigned as test teeth while the remaining tooth acted as a control site (i.e., natural colonization of the tooth surface). These teeth were then randomly assigned to receive one of the three barrier types (i.e., each patient received 4 barriers of each type, 1 per quadrant). A 2 x 5 mm piece of barrier material was positioned over the oral surface of the buccal marginal gingiva and secured with an external sling suture. With oral hygiene procedures suspended, one barrier of each type was collected at 1, 3, 7, and 14 days. Slot immunoblot assay demonstrated that all species types (A. actinomycetemcomitans, A. viscosus, B. melaninogenicus, F. nucleatum, P. gingivalis, P. intermedia, S. mutans, S. sanguis, Selenomonas sputigena, T. denticola, and T. vincentii) were present. Semi-quantitative scoring (scale 0 to 3) of slot blot results and analysis by chi-square ratio and Pearson correlation test indicated that while total bacteria adherence increased over time (P < 0.05), the 3 barrier types and the control sites did not differ in numbers or species of colonizing bacteria detected per time point. These results suggest that under these experimental conditions the barrier materials tested do not differ in bacteria adherence or antimicrobial properties.

Actinomyces viscosus↗
PubMed1997

The relationship between gingivitis and the serum antibodies to the microbiota associated with periodontal disease in children with Down's syndrome.

Gingival inflammation in Down's syndrome children (DS) develops earlier and is more rapid and extensive than in non-DS children. Abnormalities in host response to the oral flora have been proposed as etiological factors of this gingival inflammation. However, the relationship between gingivitis and the host response to oral microorganisms in DS by age has not been determined. The objective of this study was to clarify this relationship. Sera were obtained from 75 DS subjects (aged 2 to 18 years) and their gingival health assessed using a modified PMA Index (M-PMA). Antibody titers to Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Treponema denticola (Td), Fusobacterium nucleatum (Fn), Selenomonas sputigena (Sel), Actinobacillus actinomycetemcomitans (Aa), and Streptococcus mitis (Mi) were determined using the micro-ELISA. DS subjects under 4 years old were found to have significantly more gingival inflammation than did normal children the same age. A significant positive correlation (r = 0.548, P < 0.0001) existed in the relationship between M-PMA score and plaque score for subjects in the G1 age group (deciduous dentition). At G1, the average antibody titers to Aa, Mi, and Fn exceeded those of the normal adult reference serum pool. In addition, IgG antibody titers to Pg, Aa, Fn, Sel, and Mi correlated significantly with the M-PMA scores in the G1 age group. There was a correlation between age (2 to 18 years) and these antibody titers. IgG antibody titers to Pg, Aa, Sel, and Mi increased significantly with increasing M-PMA score. Furthermore, the IgG antibody titers to Pg were higher (P < 0.05) in the most extensive disease group compared to the DS no-disease group. The IgG antibody titers to Pg at G3 (early puberty) were significantly higher when compared to G1 (preschool children). The IgM antibody titers to Aa at G3 were higher (P < 0.05) when compared to G1. This study suggests that colonization by Aa and Fn are closely associated with the onset of gingival inflammation in DS patients under 5 years old. Colonization by Pg, Aa, Sel, and Mi in DS appears to be associated with gingivitis at puberty.

Adolescent↗
PubMed1996

Anaerobic microorganisms in root canals of human teeth with chronic apical periodontitis detected by indirect immunofluorescence.

Aiming to assess the presence of selected anaerobic microorganisms in root canals of human teeth with chronic apical periodontitis. 25 central and lateral upper incisors presenting with radiographic evidence of chronic apical periodontitis were studied. The pulp chamber was opened under aseptic conditions and samples of the root canal content were collected with sterile absorbent paper points, which were placed and dispersed in test tubes containing reduced transport medium RTT. Aliquots were dried on glass slides and stained by indirect immunofluorescence for detection of Actinomyces viscosus, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia. The results showed a positive indirect immunofluorescence reaction in 24 of the 25 samples. Fourteen were positive for the specie Actinomyces viscosus, 12 for Prevotella intermedia, 10 for Fusobacterium nucleatum and 4 for Porphyromonas gingivalis. A semiquantitative assay was easily implemented for assessment of degree of infection by the organisms in individual cases.

Actinomyces viscosus↗
PubMed1995

Dental findings in geriatric populations with diverse medical backgrounds.

OBJECTIVE: To determine whether there is a difference in the oral/dental health in older persons with different life styles and medical status. STUDY DESIGN: Survey (cross-sectional study) included four groups: (1) subjects (n = 123) living in a residential retirement home or community dwelling; (2) subjects (n = 218) seeking dental treatment at a Veterans Affairs Dental Outpatient Clinic; (3) subjects (n = 132) resident in a VA long-term care facility; and (4) subjects (n = 81) recently admitted to a VA acute care ward with a diagnosis of cerebral vascular accident or other neurologic problem. Each subject answered questions on medical and dental health and dietary preferences in a comprehensive interview. They were given a comprehensive dental examination that included measurements of stimulated salivary flow and minor salivary gland output. RESULTS: The data from groups 2 and 3 confirmed previous reports that independent living subjects have better oral/dental health than dependent living subjects. The data from groups 1 and 4, obtained from geriatric populations on the opposite ends of the medical health/disease continuum provide new information that suggests that good medical health and good oral/dental health are linked. The subjects in group 1 were very healthy as judged by their longevity; 54% were > or = 80 years and they had low reported prevalence of medical disease. Only 6% were edentulous and the dentate persons were missing 4.5 teeth. In contrast, over 50% of the patients in group 4 were < 70 years; they had an edentulous rate of 49% and among the dentate persons had an average 12 missing and 5 decayed teeth. CONCLUSIONS: The medically healthy persons had excellent dental health whereas the sickest persons were either edentulous or had many missing teeth.

Acute Disease↗
PubMed1995

The relationship between dental caries in the primary dentition and anti S. mutans serum antibodies in children with Down's syndrome.

The purpose of this study was to evaluate the status of dental caries in seventy-five subjects with Down's Syndrome, ages two to eighteen years and to evaluate the relationship between dental plaque or caries experience and serum antibody titers against S. mutans and S. mitis. These antibodies were examined since an enhanced host response to S. mutans might be responsible for the low caries prevalence in Down's Syndrome. Antibody titers were determined using the enzyme-linked immunosorbant assay (ELISA) and titers were expressed in ELISA units. The frequency of all subjects who were caries-free was 46.1%, while 61.4% of the subjects under five years were caries-free. The average DMFT score in the subjects of Hellman Dental Age group I (primary dentition) was 15.9. The highest frequency of caries occurred on occlusal surfaces, followed by proximal surfaces of the upper incisors and smooth surfaces on incisors. The severity of dental caries in the subjects with Down's Syndrome was bipolar and could be categorized as either mild or severe. In the subjects having a primary dentition, a significant positive correlation was found in the relationship between the Original Caries Severity Score (OCSS) and the plaque score. There was also a significant positive correlation between OCSS and IgM antibody titer to S. mutans. In contrast, no correlation between antibody titers to S. mitis and these variables was found. It is not clear if these antibodies are protective and responsible for the reduced caries rate seen in Down's Syndrome.

Adolescent↗
PubMed1993

The use of a rapid enzymatic assay in the field for the detection of infections associated with adult periodontitis.

There are few objective assays for studies of the epidemiology of periodontal diseases. The PerioScan is an assay capable of detecting three periodontal pathogens, namely T. denticola, P. gingivalis, and B. forsythus, which have been associated with adult periodontitis. The PerioScan was tested in a sample of 301 Brazilians. Clinical indices--bleeding, probing depth, gingival index, and periodontal index--were recorded from four sites in each subject. Subgingival plaque samples were collected from those sites and placed on the PerioScan card. Color results were scored in the field after 15 minutes. The plaque samples were screened with polyclonal antibodies for the three species by an ELISA system. The PerioScan, when compared with the ELISA system, yields a sensitivity of 91 percent, specificity of 89 percent, and an accuracy of 90 percent. When the PerioScan was compared to clinical indices, there was a high sensitivity (at least 93%) and a low specificity (no less than 47%), with an accuracy of at least 61 percent.

Adolescent↗
PubMed1992

Avidity and titer of immunoglobulin G subclasses to Porphyromonas gingivalis in adult periodontitis patients.

The relative avidity and titer of antibodies representing the 4 immunoglobulin G (IgG) subclasses (IgG1-4) reactive with Porphyromonas gingivalis, P. gingivalis-lipopolysaccharide (-LPS), streptokinase (SK) and tetanus toxoid (TT) in the sera of patients having adult periodontitis and of healthy controls were measured. Patient antibody titers to P. gingivalis and P. gingivalis-LPS were found to be significantly elevated for IgG, IgG1 (no P. gingivalis-LPS antibodies) and IgG2. The predominant antibody response to P. gingivalis and P. gingivalis-LPS occurred in the IgG2 subclass. When the relative avidity of the antibodies to P. gingivalis and P. gingivalis-LPS were examined, no significant differences between control and patient sera could be identified. However, anti-P. gingivalis and P. gingivalis-LPS antibodies were found to possess significantly lower relative avidity than either SK or TT antibodies. The IgG1 subclass antibodies to P. gingivalis, SK and TT all appeared to be of high relative avidity. In contrast, anti-P. gingivalis and P. gingivalis-LPS of the IgG2 subclass were of significantly lower relative avidity. Since the predominant humoral response to P. gingivalis occurs in the IgG2 subclass, the low relative avidity of these antibodies predominates in measurements of whole serum activity.

Adult↗
PubMed1992

Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary reference standard?

The development of diagnostic tests for a periodontal infection raises the issue as to what the appropriate reference standard, or "gold standard," should be for the evaluation of a new test. The present research was initiated to compare the ability of several detection methods, i.e., a serial dilution anaerobic culture and/or microscopic procedure, a DNA probe procedure, and immunological reagents using both an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay to detect Treponema denticola, Porphyromonas gingivalis, Bacteroides forsythus, and Actinobacillus actinomycetemcomitans in subgingival plaque samples taken from 204 periodontally diseased tooth sites. The prevalence of the four monitored species varied as a function of both the species and the detection method. Spirochetes were present in 99% of the plaques, whereas A. actinomycetemcomitans was detected at the lowest frequency. The culture method yielded the lowest prevalence values for the three cultivable species. This raised the question as to which results, those obtained by culture or those obtained by the DNA probes and the immunological reagents, were the most reliable. This issue was addressed by looking at the prevalence profile of the monitored organisms, as determined by all the detection methods. If the species was detected by three or four of the detection methods, then it was considered present, whereas if it was absent by three or four of the detection methods, then it was considered absent. This approach showed the DNA probes and immunological reagents to be significantly superior (P less than 0.05) to the culture approach for the detection of P. gingivalis, A. actinomycetemcomitans, and B. forsythus and to be comparable to the microscopic approach in the detection of T. denticola.

Aggregatibacter actinomycetemcomitans↗
PubMed1992

Comparison of the benzoyl-DL-arginine-naphthylamide (BANA) test, DNA probes, and immunological reagents for ability to detect anaerobic periodontal infections due to Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus.

Most forms of periodontal disease are associated with the presence or overgrowth of anaerobic species that could include Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus among others. These three organisms are among the few cultivable plaque species that can hydrolyze the synthetic trypsin substrate benzoyl-DL-arginine-naphthylamide (BANA). In turn, BANA hydrolysis by the plaque can be associated with periodontal morbidity and with the presence of these three BANA-positive organisms in the plaque. In this investigation, the results of the BANA test, which simultaneously detects one or more of these organisms, were compared with the detection of these organisms by (i) highly specific antibodies to P. gingivalis, T. denticola, and B. forsythus; (ii) whole genomic DNA probes to P. gingivalis and T. denticola; and (iii) culturing or microscopic procedures. The BANA test, the DNA probes, and an enzyme-linked immunosorbent assay or an indirect immunofluorescence assay procedure exhibited high sensitivities, i.e., 90 ot 96%, and high accuracies, i.e., 83 to 92%, in their ability to detect combinations of these organisms in over 200 subgingival plaque samples taken from the most periodontally diseased sites in 67 patients. This indicated that if P. gingivalis, T. denticola, and B. forsythus are appropriate marker organisms for an anaerobic periodontal infection, then the three detection methods are equally accurate in their ability to diagnose this infection. The same statement could not be made for the culturing approach, where accuracies of 50 to 62% were observed.

Bacteria, Anaerobic↗
PubMed1992

Application of BANA during experimental gingivitis. Application of N-alpha-benzoyl-DL-arginine 2 naphtilamide (BANA) hydrolysis to identify periodontopathic environments during experimental gingivitis in man.

This study evaluates the accuracy of the PerioScan reagent card kit which uses BANA hydrolization to detect the presence of P. gingivalis, T. denticola, and B. forsythus in dental plaque during an experimental gingivitis in man. 32 healthy subjects underwent a phase of optimal oral hygiene before they abolished all oral hygiene practices for 21 days, but rinsed twice daily with a slurry of three different toothpastes. On days 0, 7, 14, and 21, full mouth Plaque and Gingival Index scores were assessed and, in addition, on days 0 and 21, sulcular plaque samples were obtained from the mesiobuccal aspects of the second premolars. The samples were placed on BANA reagent cards (PerioScan), and the result of the trypsin-like activity read after 15 minutes. Subsequently, the samples were processed for the detection of P. gingivalis, T. denticola and B. forsythus using ELISA. The Gingival Indices on day 21 indicated a development towards gingival inflammation. The frequencies of detection of the three periodontopathogens revealed by ELISA showed increased presence of P. gingivalis, B. forsythus and T. denticola on day 21. Changes in the composition of the microbiota were also indicated by the higher rate of positive BANA results at the end of the experimental gingivitis. Without considering further clinical diagnostic tests such as "bleeding on probing", this clinically simple test does not provide a prognostic indicator for the eventual onset of disease in cases with gingival inflammation. However, specificity was only 61% and the sensitivity was 41.7%.

Adult↗
PubMed1991

Measurement of relative avidity of antibodies reactive with Porphyromonas (Bacteroides) gingivalis in the sera of subjects having adult periodontitis.

Relative avidities of antibodies to Porphyromonas (Bacteroides) gingivalis in the sera of 15 patients having adult periodontitis and 15 healthy subjects were evaluated using an ammonium thiocyanate-dissociated ELISA. Graded concentrations of ammonium thiocyanate were added to a single dilution of serum in order to dissociate low avidity antibody binding to P. gingivalis. The concentration of thiocyanate resulting in 50% reduction in binding (absorbance) was termed the ID50 for that serum. When IgG-class antibodies were examined, the ID50 of anti-P. gingivalis antibodies in the sera of patients was significantly elevated (0.96M vs 0.71M; p less than 0.01, Student's t-test). In contrast, when IgM-class antibodies were examined no significant differences in ID50 between patients and controls were found for P. gingivalis (0.54M vs 0.53M). While the ID50 values of patient antibodies were found to be elevated relative to those of healthy controls, comparison with antibodies from rabbits immunized with P. gingivalis and with ID50 values from other human studies suggests that adult humans, in general, produce very low-avidity antibodies to P. gingivalis. It is suggested that the presence of low-avidity antibodies contributes to the pathology associated with periodontal disease.

Adult↗
PubMed1991

Slot immunoblot assay for detection and quantitation of periodontal disease-associated microorganisms in dental plaque.

A rapid method for qualitative and quantitative detection of specific oral microorganisms from subgingival dental plaque is described. Plaque samples were suspended in phosphate-buffered saline containing protease inhibitors and 0.5% formaldehyde, briefly sonicated to disperse bacterial aggregates, and applied to nitrocellulose membranes in a slot blot manifold. Subsequent incubations with species-specific rabbit antibody and anti-rabbit antibody-alkaline phosphatase conjugate and development with BCIP-NBT substrate resulted in an easily discernible, permanent stain being deposited at the sample application site. Comparison with known concentrations of pure, cultured microorganisms applied to the same membranes permitted qualitative or semiquantitative plaque characterization by visual inspection. Analysis of the blots with a computer-linked flatbed scanner provided quantitative data on microbial content. The reproducibility of the results (standard error of the mean, less than 10%) obtained with slot immunoblotting greatly exceeded that of the results obtained with immunofluorescence analysis (standard error of the mean, greater than 57%). Because it is versatile, rapid, sensitive, reproducible, permanent, and relatively inexpensive, slot immunoblotting lends itself to use in large-scale investigations for the detection and quantitation of specific microbial species.

Adult↗
PubMed1991

Detection of two anaerobic periodontopathogens in children by means of the BANA and ELISA assays.

The mouths of young children become colonized by a variety of bacteria, but there have been only a few studies that have sought the presence of periodontopathic species in this population. Almost all of these studies used culturing techniques rather than the newer detection methodologies for various periodontopathogens. Studies in adults have shown that Treponema denticola and Porphyromonas (Bacteroides) gingivalis can be detected in dental plaque by use of the BANA and ELISA diagnostic tests. In the present study, plaque samples from four subgingival sites in each of 157 children (aged from two to 18 years) were tested for BANA hydrolysis with a BANA reagent card, and for T. denticola and P. gingivalis with an ELISA assay. Anaerobic periodontopathogens hydrolyzing the BANA substrate were found to be present in at least one of four plaque samples in 88 children (56%). T. denticola and/or P. gingivalis were detected by ELISA in at least one plaque sample in each of 135 children (86%). This study shows that children are widely colonized by these micro-organisms. A higher proportion of Black children than Caucasian children was colonized by these BANA-positive organisms. Also, children having a parent with a documented history of periodontal disease were more likely to be BANA-positive than were children of parents with unknown periodontal status.

Adolescent↗
PubMed1991

Periodontal healing following guided tissue regeneration with citric acid and fibronectin application.

This study was undertaken to determine the effects of guided tissue regeneration (GTR) with and without citric acid conditioning and autologous fibronectin application. The study subjects were four female beagle dogs with spontaneous periodontitis. The dogs were given thorough root debridement and 4 weeks later, mucoperiosteal flaps were raised on both sides of the mandible involving the 2nd, 3rd, and 4th premolar and 1st molar teeth. After debridement, notches were placed on the roots at the level of supporting bone. Citric acid (pH 1) was topically applied for 3 minutes on the exposed root surfaces of one side (experimental). The roots were irrigated with normal saline solution. Both the root surfaces and the inner surface of the flap were then bathed in autologous fibronectin in saline. Following this, Gore-Tex periodontal material was adapted to the roots of each tooth and sutured. The contralateral side, serving as control, was treated by surgery and application of Gore-Tex periodontal material only. All membranes were removed 1 month after surgery, and the dogs sacrificed at 3 months. Both mesio-distal and bucco-lingual microscopic histological sections were evaluated by descriptive histology, and linear measurements and surface area determination of the furcal tissues were made. Periodontal healing following the use of GTR procedure resulted in an increase in connective tissue and alveolar bone regeneration. Adjunctive critic acid plus autologous fibronectin produced slightly better results, but these differences were not statistically significant for this sample.

Acid Etching, Dental↗
PubMed1991

Use of BANA hydrolysis as a diagnostic test for identifying periodontopathic environments.

The purpose of the present study was to evaluate the diagnostic value of the BANA hydrolysis test using the Perio Scan kit to identify subgingival periodontopathogens (P. gingivalis, T. denticola and B. forsythus) and to determine whether the BANA reaction reflected the presence or absence of clinical signs for periodontitis. 10 patients with periodontitis (AP), 10 healthy subjects (HS) and 10 subjects with treated periodontitis under supportive therapy (MP) participated in the study. Subgingival plaque was sampled and the presence or absence of the above mentioned bacteria assessed with BANA reagent cards (Perio Scan). Additionally, all samples were analyzed for the presence or absence of P. gingivalis and/or T. denticola using an ELISA. The BANA test yielded a high specificity (86.7%) and a high sensitivity (77.0%) for the presence of periodontopathogens when compared with the ELISA results as reference parameter. The diagnostic features of the BANA test result compared to the clinical parameters yielded a high degree of accuracy for overall clinical judgement and for probing depth (PD 5 mm), but to a lesser degree, for bleeding on probing.

Adult↗
PubMed1990

Benzoyl-arginine naphthylamide (BANA) hydrolysis by Treponema denticola and/or Bacteroides gingivalis in periodontal plaques.

Treponema denticola and Bacteroides gingivalis are among the few recognized species found in periodontal pockets that can hydrolyze the synthetic peptide N-benzoyl-DL-arginine-2-naphthylamide (BANA). We determined the presence of these periodontal pathogens in BANA-positive and -negative plaque samples through the use of indirect immunofluorescent antibody techniques. Eighteen of 27 diseased sites gave BANA-positive reactions, and 9 gave BANA-negative reactions. T. denticola was present in 16 of 18 BANA-positive reactions, whereas B. gingivalis was detected in 9 of the 18 BANA-positive reactions. T. denticola was present in 1 and B. gingivalis in 2 of the 9 BANA-negative reactions. Neither organism was detected in the 19 healthy sites that were negative for BANA. All measured differences between BANA-positive and BANA-negative plaques obtained in the same individuals were statistically significant. The accuracy of the BANA test, compared with clinical parameters such as bleeding upon probing and increased probing depth, was about 80%. The accuracy of the test in detecting the presence of T. denticola was 93%, for B. gingivalis, 76% and for T. denticola and/or B. gingivalis, 96%. This study indicated that BANA-positive plaques were associated with the presence of T. denticola and/or B. gingivalis, that T. denticola was found at a greater frequency and levels in BANA-positive plaques than B. gingivalis, and that the presence of these organisms was associated with clinical disease.

Adult↗